6alpha, 16alpha-dialkyl steroids and intermediates



Patented Oct. 17, 1967 droxy-S-pregnen-ZO-one 3-tosy1ate 21-acylate which has th 1 3 347,881 e fol owing structural formula 6u,16a-DIALKYL STEROIDS AND 011,011

INTERNEDIATES Meyer Sletzinger and Donald F. Reinhold, North Plainf field, N.J., assignors to Merck & (30., Inc., Rahway,

N.J., a corporation of New Jersey No Drawing. Original application Sept. 12, 1958, Ser. No.

760,562, new Patent No. 3,094,523, dated June 18,

1963. Divided and this application July 16, 1962, Ser. 10

3 Claims. (Cl. 260-397.47)

This invention is concerned generally with steroid comtosyl'o pounds and with novel proceses of preparing the same. 1 wherein R and are as above. More particularly, it relates to 6u,16a-dialkyl-11-oxy- The 16 1k 1 3 3 1'7 21 t ih d 5 2Q -v genated Steroids 0f the Pregnant? Series unsaturated in 3-tosylate 21- acylate is reacted with methyl ethyl ketone ring A and to a novel process and intermediates produced d potassium t t t form 16a-alkyl-3,5-cycl0-6fl,

in P P compounds- 17a,21-trihydroXy-20-pregnanone 21- acylate which may This is a dlVlSlOll of co-pendrng applicatlon, Ser. No. be represented as follows;

760,562, filed Sept. 12, 1958, now United States Patent No. 3,094,523. CHzOR These 6a,16 x-dialkyl-1l-oxygenated steroid compounds possess extremely high anti-inflammatory activity, and are ---OH especially effective for the treatment of arthritis and re- 25 lated diseases since they can be administered for their cortisone-like action in extremely low dosage thereby minimizing undesired side effects.

In preparing these active compounds, the starting material utilized is 16a-alkyl-5,6-dihalo-3fi,17a,21-trihydr0xy- 20-pregnanone 21-acylate which may be represented graphically as follows:

16a-alkyl-3,5-cyclo-6;8,17a,21-trihydroXy-20 pregnanone CHQOR' 21-acylate is reacted with an oxidizing agent such as i chromium trioxide to form 16a-alkyl-3,5-cyclo-17a,2l-dihydroxy-6,20-pregnanedione 21-acy1ate which has the following structural formula:

40 (llHzoR y HO 7 I X it I wherein R is alkyl, R is acyl and X is halogen. The H 0 above 16a-alkyl-5,6-dihalo-3,8,170:,2l-trihydroxy-ZO-preg- 5Q nanone 21-acylate is reacted with chromous halide to form 16a-alkyl-3fi,17,21-trihydroxy-5-pregnen-20-one 21-acylwherein R and R are as above. ate which may be indicated graphically as follows: The 16u-alkyll-3,5-cyclo-17a,21-dihydroxy- 6,20-pregnanedione 2l-acylate is hydrolyzed with sodium methoxide to form the 21-alcohol, namely, l6u-alkyl-3,5-cyclo- 17a,21-dihydroxy-6,20-pregnanedione which may be rep 0:0 resented as follows:

Q its CHzOH wherein R and R are as above.

nen-ZO-one 21-acy1ate with para-toluenesulfonyl halide The 1601. alkyl 3,5 cyclo 17:2,21 dihydroxy-6,20- results in the formation of 16a-alkyl-3B,17a,21-trihypregnanedione is reacted with formaldehyde to form 1611- I 3 alkyl 3,5 cyclo l7a,20,20,21 bismethylenedioxy 6- pregnanone which has the structural formula:

wherein R is as above.

Treatment of the above l6a-alkyl-3,5-cyclo-l7u,20,20, 21-bismethylenedioxy-6-pregnanone with an alkyl magnesium halide yields a mixture of isomers of 6e,l6a-dialkyl- 3,5 cyclo 6e hydroxy 17o,20,20,2l bisrnethylenedioxypregnane which may be represented as follows:

to Q i wherein R and R are as above.

The 6,16u dialkyl 3B hydroxy 1704,20,20,21 bismethylenedioxy-S-pregnene 3/3-acylate is hydrolyzed to 6,160: dialkyl 3/3 hydroxy 17a,2(),20,21 bismethylenedioxy-S-pregnene which can be represented as follows:

wherein R is as above.

4 The 6,16u dialkyl 3,8 hydroxy l7cc,2(i,20,2l bismethylenedioxy-S-pregnene can be reacted with cyclohexanone and aluminum isopropoxide to form ,16adialkyl 17u,20,2(),2l bismethylenedioxy 4 pregnen- 3-one which has the following structural formula:

wherein R is as above.

Treatment of the above 6a,l6a-dialkyl-17a,20,20,2lbismethylenedioxy-4-pregnen-3-one with acid results in the formation of 60:,l6a dialkyl 1704,21 dihydroxy-4- pregnene-3,20-dione which may be indicated as follows:

CHaOH wherein R is as above.

The 6a,16ot dialkyl 17:1,21 dihydroxy 4-pregnene- 3,20-dione can be subjected to the action of an oxygenating enzyme produced by a strain of Curvularia lunaza to from 611,160: dialkyl-l1p ,17a,21-trihydroxyl-4-pregnene- 3,20-dione, a compound which possesses high anti-inflammatory activity and is especially effective in the treatment of arthritis and related diseases. The organism Curvularia lunata can be obtained from known sources such as the Northern Regional Research Laboratory (N0. 2434), Peoria, Ill. or they may be isolated from natural sources, such as soil, by known methods.

The 6a,16a-dialkyl-1lfl,17vc,21 trihydroxy-4-pregnene obtained can be treated with acetic auhydride to form the 21-acylate derivative which is purified by recrystallization from benzene petroleum ether to form substantialy pure 60:,16ot-dl3lliYl-l1fl,17a,21 trihydroxy-4-pregnene-3,2()-dione 21-acylate.

The hydroxy group in the llfl-position can be converted to ll-keto by oxidation of 6a,16ocdialkyl-11fi,17a, 21-trihydroxy 4 pregnene-3,20dione 21-acylate with chromium trioxide in the presence of pyridine to form 6a,16u-dialkyl-l7a,2l-dihydroxy 4 pregnene-3,1 1,20- trione 21-acylate. The 6a,16a-dialkyl 1lfi,17oc,21 trihydroxy 4 pregnene-3,20-dione 2l-acylate esters thereof and 6a,16a-dialky1-l7a,2l-dihydroxy 4 pregnene-3,11, 20-trione and 21-acy1ate esters thereof have been found to possess extremely effective anti-inflammatory activity in the treatment of arthritis and related diseases.

Alternatively, 6a,16a-dia1 kyl-17a,21dihydroxy-4-pregneue-3,20-di0ne can be subjected to the action of an oxygenating enzyme produced by a strain of Rhizopus nigricans to introduce a lla-hydroxy group into the steroid thus forming 6a,16u-dialkyl-11a,l7a,2l-trihydroxy- 4-pregnene-3,20-dione. The Rhizopus nigricans organism can be obtained from known sources such as the American Type Culture Collection No. 6227b, Washington, DC, or they may be isolated from natural sources, such as soil; by known methods. Oxidation of the lloz-hYdIOXY group to ll-keto can be accomplished by a reaction with chromous acid to form 6a,16a-dialkyl-17u,21-dihydroxy- 4-pregnene-3,11,20-trione. The 3,20-positions of the ring can be protected by the introduction of semicarbazone groups. This procedure involves reacting 6u,l6a-dialkyl- 17a,21-dihydroxy 4 pregnene-3,l 1,20-trione with semicarbazide halide to form 3,20-disemicarbazone of 6a,16 xdialkyl-17a,2l-dihydroxy-4-pregnene-3,11,20-trione. This latter compound is then reacted with sodium borohydride in tetrahydrofuran to introduce the hydroxy in the 115- position. The 3,20-bis-semicarbazone groups can be removed by treatment with pyruvic acid to form 60:,16ocdialkyl-l 1p,17u,21-trihydroxy-4pregnene-3,20-dione.

The 6a,l6tx-dialkyl l113,17a,2l-trihydroxy-4-pregnene- 3,20-dione or 6a,l6a-alkyl-dialkyl 17a,21-dihydroxy-4- pregnene-3,ll,20-trione can be reacted with selenium dioxide to introduce the double bond in ring A at the l-position thereby forming 6c:,16a-dlalkyll1fl,17oc,2l-tllhydroxy-1,4-pregnadiene 3,20-dioneor 6u,l6a-dialkyl- 17u,2l-dihydroxy-1,4-pregnadiene-3,11,20-trione.

Alternatively, the double bond can be introduced into a 611,1 6a-dialkyl-17a,2 1-dihydroxy-4-pregnene-3,ZO-dione employing selenium dioxide thus forming 6a,l6a-dialkyl- 17u,21-dihydroxy-1,4 pregnadiene-3,20-dione 21-acylate. The llfi-hydroxy-group can be introduced into the latter compound employing Curvularia lunata to form 6a,l6adialkyl-l1fl,17a,21-trihydroxy 1,4 pregnadiene3,20- dione. It is also possible to introduce the lla-hydroxy into the 641,16u-dialkyl-l7a,2l-dihydroxy 1,4 pregnadiene- 3,20-dione 21-acylate, oxidizing the lla-hydroxy to the ll-keto compound, protecting the 3 and 20-position with semicarbazone groups and introducing the llp-hydroxy. The 3,20-semicarbazone can be removed leaving 60:,l6ocdialkyl-llB,17a,21-trihydroxy 1,4 pregnadiene-3,20- dione.

The 6a,16a-dialkyl 115,17a,21 trihydroxy-1,4-pregnadiene-3,20-dione and 21-acylate esters and the 6m,16adialkyl-17,2l-dihydroxy-1,4-pregnadiene-3,20-dione and 21-acylate esters obtained by these procedures possess extremely high anti-inflammatory activity and are especially effective in the treatment of arthritis.

The 16u-alkyl-5,6-dihalo-3;8, l7a,2l-trihydroxy-20-pregnanone 21-acylate used as starting materials in this process are conveniently prepared starting with the known 3- acyloxy-S,16-pregnadien-20-one in accordance with the following procedure:

3-acyloxy-5,16-pregnadien-20-one is reacted with alkyl magnesium halide thereby forming 16a-alkyl-3B-hydroxy- 5-pregnen-20-one which is reacted with an acyl anhydride in the presence of pyridine to form 3B-acy1oxy-16aalkyl-S-pregnen-ZO-one. The latter compound is reacted with halogen to form 3B-acyloxy-16a-alkyl-5,6-dihalo-20- pregnanone, which is then reacted with a dialkyl oxalate. The resulting alkyl ester of 16a-alkyl-5,6-dihalo-3fl-hydroxy-20-oxo ZI-pregnane-glyoxylic acid is hydrolyzed With an alkali metal hydroxide to form 16a-alkyl-5,6-dihalo-3,8-hydroxy-20-oxo-2l-pregnane-glyoxylic acid. The latter compound is reacted with nitrobenzene sulfonic acid and an acyl anhydride to form 3 13,23-diacyloxy-16aalkyl-5,6-dihalo-2l-normethyl 17(20),22 choladieno- 24(20)-lactone. Reaction of the latter compound with a peracid yields 3,9,23-diacyloxy-16a-alkyl 5,6-dihalo-21- normethyl-17(20)-oxido 22 cholano-24(20)-lactone which is reacted with base to yield l6a-alkyl-5,6-dihalo- 3 3,17a-dihydroxy-20-pregnanone. The latter compound is reacted with a halogenating agent to form 16a-alkyl-5,6, 2l-trihalo-3fl,l7ot-dihydroxy-20-pregnanone. Reaction of the latter compound with an acylating agent results in the formation of 16a-alkyl-5,6dihalo 3,8,17a,21-trihydroxy- 3,20-pregnanedione 2l-acylate.

In all of the previously described reactions, R has been designated as alkyl, and is intended to include the lower alkyl groups such as methyl, ethyl and propyl or aryl alkyl group. R has been indicated as acyloxy. The

acyloxy groups that may be employed in these reactions include lower hydrocarbon carbonyloxy esters such as benzoate, lower alkanoates such as acetoxy, and propionoxy, X is halogen and is intended to include bromo and chloro. It will be evident to those skilled in the art that other groups may be substituted for those recited here and these definitions are intended only as some indication of the operable class of compounds.

The following experimental part illustrates in detail some of the compounds which constitute this invention and methods for their production. However, this invention is not to be construed as limited thereby in spirit or in scope which it will be apparent to those skilled in the art that many modifications in materials and methods may be made without departing from the invention.

EXAMPLE 1 Preparation of 3 8,1 7 (1,21 -trihydr0xy-I 6 ot-methy l-5 pregnen-ZO-one 21 -acetaze A 3 ml. aqueous acetone solution of chromous chloride, prepared by reduction of chromic chloride with amalgamated zinc, is added to .13 g. of 5,6-dichloro-3fi, 1705,21 trihydroxy-l6a-methyl-20-pregnanone 2l-acetate (prepared as described herein below). The mixture is refluxed for five minutes under nitrogen, then cooled and diluted with 5 ml. of water. The precipitate which separated from solution, is filtered and recrystallized from acetone to yield mg. of 3B,17u,21-trihydroxy-16umethyl-S-pregnen-ZO-one 21-acetate which melted at 185- 188 C.

Analysis.-Calculated for C H O (404.53): C, 71.26; H, 8.97. Found: C, 70.95; H, 9.28.

The 5,6-dichlo1o-3fi, 17a,21-trihydroxy-1 6cc-Hlfitl1Yl-20- pregnanone 21-acetate used as a starting material in this example is prepared as follows:

A solution of 10 g. of methyl iodide in 40 ml. of ether is added to 1.73 g. of magnesium in 50 ml. of ether. To the resulting ml. of ethereal solution of methyl magnesium iodide, maintained under a nitrogen atmosphere is added 50 ml. of ether followed by the addition of .64 g. of cuprous chloride. The mixture is stirred vigorously for 1.5 hours at 20 C. and then refluxed for /2 hour. At the end of this time the yellow cuprous chloride is converted to a fine black insoluble powder. A solution of 16.4 g. of 3-acetoxy-5,16-pregnadiene-20- one in 500 ml. of absolute ether, is added over a period of 1 hour. The mixture is then refluxed for 6 hours with stirring. The methyl magnesium iodide complex and excess methyl magnesium iodide is decomposed by the careful addition of ml. of 10% sulfuric acid. The mixture is then filtered through diatornaceous earth and transferred to a separatory funnel. The aqueous acetic layer is separated and back extracted with 200 ml. of ethyl acetate. The ethyl acetate is combined With the ether layer and washed successively with water, 5% sodium sulfite solution, water, 10% sodium bicarbonate solution and finally with water. The organic solution is dried and concentrated in vacuo until crystallization begins. The ethyl acetate solution is then distilled at atmospheric pressure until crystallization of 3fl-hydroxy l6amethyl-S-pregnene-ZO-one begins. The solution is cooled to 10 C. and filtered. Wt. 10.89 g. M.P. 182-188 C Recrystallization from ethyl acetate raises the melting point to 189191 C.

A mixture of 50.0 g. of 3 8-hydroxy-16a-methyl-5- pregnene-ZO-one in 200 ml. of acetic anhydride and 200 ml. of pyridine is allowed to stand for 18 hours at 20 C. The mixture is concentrated to dryness and the residue dissolved in benzene. The benzene solution is washed with 25% sulfuric acid, saturated sodium bicarbonate solution and water. The benzene solution is dried and concentrated to dryness. Petroleum ether is added to induce crystallization. The

pregnen-ZO-one is filtered and dried. Wt. 50.3 g. M.P. 181-185 C.

Chlorine gas is slowly bubbled into 40 ml. of benzene containing .21 ml. of pyridine. Simultaneously, a solution of 6.0 g. of 3,8-acetoxy-16ut-methyl-5-pregnen-20-one in .40 m1. of benzene containing .21 ml. of pyridine is added to the chlorine-benzene solution. The rate of addition of the steroid solution is adjusted so that a slight excess of chlorine is present. The addition of the steroid requires 25 minutes. The reaction mixture is poured into sodium thiosulfate solution to destroy the excess chlorine. The benzene solution is washed with 5% hydrochloric acid, sodium bicarbonate solution and water. The benzene solution is then dried and concentrated to dryness. The residue is dissolved in hot acetone, treated with charcoal and filtered. The filtrate is evaporated on a steam bath until crystallization starts. The mixture is cooled and filtered to yield 3fl-acetoxy-5,6-dichloro-16amethyl-20-pregnanone which melts at 195 -198 C.

Sodium methoxide is prepared by adding 35 ml. of methanol to 3.18 g. of clean sodium in a 250 ml. roundbottom flask. To the mixture is added 100 ml. of dry toluene and the mixture is distilled slowly until the boiling point of the distillate is 110 C. Diethyl oxalate (24.6 grams) is then added slowly to the cooled sodium methoxide solution maintaining the temperature between 20-25 C. Then 11.8 grams of 3B-acetoxy-5,6-dichloro- 16a-methyl-20-pregnan0ne is added and the yellow solution stirred for 18 hours.

The mixture is poured slowly with stirring into 250 ml. of ether-petroleum ether (1:1) to precipitate the sodium salt of the ethyl ester of 5,6-d1ChlOIO-3j3-hYClI'OXY-16amethyl-20-oxo ZI-pregnaneglyoxylic acid which is collected by filtration. Ethanol (110 ml.) is then added to the sodium salt of the ethyl ester of 5,6-dichloro-3p-hydroxy- 16m methyl-20-oxo-21-pregnaneglyoxylic acid and the mixture acidified by addition of 2.5 N hydrochloric acid. To this mixture is added 250 ml. of water and 250 ml. of benzene and stirred for minutes. The benzene layer is separated and washed with Water until neutral. The benzene solution is dried and concentrated in vacuo to yield 16.3 g. of the ethyl ester of 5,6-dichloro-3fi-hydroxy- 16u-methyl-20-oxo-21-pregnaneglyoxylic acid U.V. k 2920, E% 135.

To 16.3 grams of the ethyl ester of 5,6-dichloro-3B- hydroxy-16ot-methyl-20-oxo-2l-pregnaneglyoxylic acid is added 250 ml. of methanol and then over a period of minutes 250 ml. of .5 N sodium hydroxide is added. The mixture is stirred for 5 hours and then rapidly heated to 70 C. and held at 70 C. for five minutes. The clear solution is cooled rapidly to C. and acidified with 2.5 N hydrochloric acid. The precipitate is collected and washed thoroughly with Water. The crude 5,6-dichloro-3B-hydroxy 16a methyl-20-oxo-2l-pregnaneglyoxylic acid is slurried with 100 ml. of acetonitrile, filtered and dried. Wt. 10.02 grams, M.P. 209211 C. U.V. A 2910, E% 208.

Acetic anhydride (82 ml.) is added to 2.2 grams of 5,6-dichloro-3/3-hydroxy-16a-methyl-20-oxo-2l-pregnaneglyoxylic acid. To this suspension is added 300 mg. of dinitrobenzene sulfonic acid. The mixture is stirred for 2 hours and 300 mg. of potassium acetate is added and the solution concentrated in vacuo below C. to dryness. The 36,23-diacetoxy 5,6 dichloro-16a-methyl-2l-normethyl-17(20),22-choladieno-24(20)-lactone is dissolved in 250 ml. of benzene and washed successively with two portions of ml. of cold 2.5 sodium hydroxide and two portions of 100 cc. of water. The benzene solution is dried and concentrated to dryness to yield an orange-red oil which yields light tan crystals on trituration with ethanol. The 35,23-diacetoxy-5,6-dichloro-16a-methyl-21- normethyl 17(20),22-cho1adieno-24(20)-lactone weighs 8.6 g. U.V. 1 2990, E% 423.

A solution of 8.61 grams of 3fl,23-diacetoxy-5,6-dichloro-16e-methyl-21-normethyl-17(20),22 choladieno- 24(20)-1actone is dissolved in 82 ml. of .55 M perbenzoic acid in benzene. The solution is allowed to stand at room temperature for 5 days. The benzene solution is then washed successively with 5% sodium bisulfite solution, saturated sodium bicarbonate solution, and finally with saturated sodium chloride solution. The benzene solution is dried and concentrated to dryness to yield an oil which crystallizes on addition or" 50 ml. of petroleum ether. Filtration yields 8.68 grams of 313,23 diacetoxy-5,6-dichloro-16a-methyl-21-normethyl 17(20) oxido-22-choleno-24(20)-lactone U.V. A 2280, E% 194.

A solution of .5 g. of 3/3,23-diacetoxy-5,6-dichloro- 16a-methyl-21-normethyl-17(20)-oxido 22 choleno- 24(20)-lactone in 6 ml. of tetrahydrofuran and 20 ml. of 2.5 N sodium hydroxide is stirred for 20 hours at room temperature. The tetrahydrofuran layer is separated and water added to precipitate 5,6-dichloro-3B,17u-dihydroxy- 16a-methyl-20-pregnanone. This product is filtered and the cake dissolved in ethyl acetate. The ethyl acetate solution is washed with sodium bicarbonate solution and water. Concentration of the ethyl acetate yields 300 mg. of 5,6-dichloro 313,170 dihydroxy-16a-methyl-20-pregnanone. M.P. 203-209 C. (dec.). Recrystallization from methanol raises the melting point to 215-216 C. dec.

To a solution of .207 gram of 5,6-dichloro-3B,17u-di hydroxy-l6a-methyl-20-pregnanone in 5 ml. of chloroform containing .01 ml. of methanol is added over a period of 1 hour, 1.56 ml. of .352 M bromine in chloroform solution. The chloroform solution is poured into ether and washed successively with saturated sodium bicarbonate solution and water. The chloroform-ether solution is dried and concentrated to yield an oil which crystallizes on standing. This crystalline material, namely 21- bromo 5,6 dichloro-3t3,17a-dihydroxy-16a-methyl-20- pregnanone is recrystallized from benzene-petroleum I ether. M.P. -194 C.

EXAMPLE 2 Preparation of 35,1 7a,21-trihydroxy-16a-methyl-5- pregnen-ZO-one-S-tosylate 21 -acetale To a solution of 50 mg. of 3B,l7ot,2l-lflhYdfOXY-lfiamethyl-S-pregnen-ZO-one 21-acetate in 5 ml. of pyridine is added 35 mg. of p-toluenesulfonylchloride. The mixture is allowed to stand for sixteen hours at about 20 C. The

' mixture is then diluted with water and extracted with chloroform. The chloroform extract is washed with four 3 ml. portions of water. The chloroform solution is dried and concentrated to dryness. The residue is dissolved in acetone and petroleum ether is added until the solution is turbid. After standing for about fifteen hours, 35,1704, 21-trihydroxy-16u-methyl-S-pregnen-20-one 3-tosylate 21- acetate crystallizes from solution. The mixture is filtered, washed with petroleum ether and dried. M.P. 176-178 C.

EXAMPLE 3 Preparation of 3,5-cycl0-6B,16a,21-trihydr0xy16amet/1yI-ZO-pregnanone 21 -acetate A mixture of 16.6 mg. of 3[i,17a,2l-trihydroxy-16amethyl-S-pregnen-ZO-one 3-tosylate 21-acetate, 1 ml. of methyl ethyl ketone, 21 mg. of potassium acetate and .2 ml. of water is refluxed for 18 hours. The methyl ethyl ketone is evaporated in a stream of nitrogen. The residue is triturated with water and dissolved in a mixture of benzene-ethyl acetate. The organic layer is washed with saturated sodium bicarbonate solution and water, dried and concentrated to dryness to yield an oil.

x533; ass-2.97 5.74 (sh), 5,81,., 5.91,.

Analysis.Calculated for C24H3 05.C3H 0: C, H, 9.15. Found: C, 69.80; H, 8.84.

EXAMPLE 4 A solution of 50 mg. of 3,5-cyclo-6B,l7a,2l-trihydroxy- 16a-methyl-20-pregnanone 2l-acetate in .5 ml. of pyridine is prepared. A solution of 50 mg. of chromium trioxide in .5 ml. of pyridine is then added and the mixture allowed to stand for about fifteen hours at 20 C. The reaction mixture is diluted with hot benzene and filtered through diatomaceous earth. The benzene filtrate is washed successively with l N hydrochloric acid, water, sodium bicarbonate and water. The benzene solution is dried, filtered and concentrated to dryness. The residue is crystallized from ethyl acetate-petroleum ether to yield 3,5- cyclo-l7ot,21-dihydroxy 16a methyl 6,20 pregnanedione 21 -acetate.

CHO13 mart.

Preparation 5.74 1, (sh), 5.79a, 5.95;

EXAMPLE 5 Preparation of 3 ,5 -cycl 0-1 7a,21-dihya'roxy-1 6 (X- methyl-6,20-pregnanedione To a solution of 363 mg. of 3,5-cyclo-17a,2l-dihydroxyl6a-methyl-6,ZO-pregnanedione 21-acetate in 20 ml. of purified methanol is added a .42 ml. solution of 2.16 N sodium methoxide in methanol. The solution is stirred for 8 minutes and then is made slightly acid with acetic acid. The mixture is diluted with water and the precipitate is collected by filtration. The hydrolyzed product 3,5- cyclol7a,2l dihydroxy 16oz methyl 6,20-pregnanedione is washed thoroughly with water and is employed directly in the next reaction.

EXAMPLE 6 Preparation 0 3,5 -cycl0-1 6 a-methy l-l 70,2 0,20,21 ibism ethylenedioxy-o-pregnanone To a mixture of .280 g. of 3,5-cyclo-17a, 21-dihydroxy- 16u-methyl-6,20-pregnanedione in 11 ml. of chloroform is added a solution of 2.8 ml. of concentrated hydrochloric-acid and low methanol 37% formaldehyde, premixed at 0 C. The two phase system is stirred at 20 C. under nitrogen for 24 hours. The chloroform layer is then separated and the aqueous layer is extracted twice with 25 ml. portions of chloroform. The chloroform extracts are combined and washed with saturated sodium bicarbonate solution and water. The chloroform solution is dried and concentrated to dryness. The residue is flushed twice with 20 ml. portions of methanol. The residue is then dissolved in benzene and chromatographed on Florosil. The column is eluted successively with 1%, 3%, 5% and 7.5% acetone in n-hexane. The crystalline fractions from the 5% acetone cuts, which give a negative blue tetrazolium test are combined and recrystallized from methanol to yield substantially pure 3,5-cyclo-16a-methyl- 17a,20,20,2l-bismethylenedioxy-6-pregnanone.

hydroxy 66,160; dimethyl 17a,20,20,21 bismethylenedioxypregnaize To a solution of 150 mg. of 3,5-cyclo-l6a-methyl-l7a, 20,20,21-bismethylenedioxy-6-pregnanone in 25 ml. of

ether is added dropwise a solution of 1 ml. of 3 M methyl magnesium in ether. The mixture is refluxed for two hours, cooled and excess Grignard reagent is decom posed with a saturated ammonium chloride solution. The ether layer is separated and the aqueous layer is extracted with chloroform. The organic extracts are combined, washed with water, dried and concentrated. The residue which is probably a mixture of isomers of 3,5-cyclo-6ehydroxy 6e,l6ot dimethyl-17a,20,20,21-bismethylenedioxypregnane is used in the next stepwithout purification.

EXAMPLE 8 Preparation of 3 p-hydroxy-lil 6 a-dimethyl-l 711,2 0,20,21 bismethylenedioxy-S-pregnen-3/3-acetate A solution of 150 mg. of a mixture of isomers of 3,5- cyclo 6e hydroxy 66,1611 -dimethyl-17a,20,20,2l-bismethylenedioxypregnane in 2 ml. of 10% sulfuric acid in acetic acid is stirred for about fifteen hours at 20 C. The mixture is diluted with ice Water and the solids collected by filtration. The cake is washed thoroughly with water and dried. The product, is 3fi-hydroxy-6,l6a dimethyl 17oc,20,20,21 bismethylenedioxy- 5-pregnen- 3,6-acetate.

EXAMPLE 9 Preparation 0 f 3 3-hydroxy-6J 6 a-dimethy l-] 7 a,2 0,2 0,2 1 bismethy lenedioxy-S-pregn ene The 3fi-hydroxy 6,160: dimethyl 17a,20,20,21 bismethylenedioXy-S-pregnen-3fi-acetate obtained in the previous Example 8 is hydrolyzed by dissolving it in 5 ml. of methanol, adding 5-0 mg. of potassium carbonate and refluxing on the steam bath for one hour. The mixture is cooled, diluted with 1 ml. of Water and the methanol evaporated in a stream of nitrogen. The precipitate is filtered, washed thoroughly with water and recrystallized from aqueous methanol to give 3B-hydroxy-6-16adimethyl-17a,20,20,2l-bismethylenedioxy-S-pregnene.

EXAMPLE 10 Preparation of 6a,16o.--dimethyl-]7u,20,20,21-bismethyZ-. enedioxy-4-pregnen-3-one To a solution of 5 ml. of toluene and mg. of 6,16adimethyl-17a,20,20,2l-bismethylenedioxy-5-pregnene is added .25 ml. of cyclohexanone and mg. of redistilled aluminum isopropoxide. The mixture is heated under reflux for 45 minutes. A .25 ml. solution of potassium sodium tartrate is added and the mixture is steam distilled to remove the toluene and cyclohexanone. After cooling, the aqueous suspension is extracted with chloroform. Concentration of the chloroform in vacuo yields an oil which is triturated with petroleum ether to remove traces of cyclohexanone. The residue is dried thoroughly in vacuo and is crystallized from benzene-heptane to give 6a,l6ot dimethyl 17oc,20,20,2l bismethylenedioxy 4- pregnen-3-one.

EXAMPLE 11 Preparation of 1 70,21-dihyar0xy-6a,16a-dimethyl-4-pregnene-3,20-di0ne A solution of 70 mg. 6a,16a-dimethyl17a,20,20,21- bismethylenedioxy-4-pregnen-3-one in 7 ml. of 50 acetic acid is heated on a steam bath under nitrogen for. 8 hours/The solvents are concentrated to dryness in. vacuo. The residue is dissolved in chloroform and washed with a saturated sodium bicarbonate solution. The chloro-v form solution is filtered and concentrated to a small volume. The chloroform solution is then streaked on paper strips. The paper is dipped in a formamide methanol solution (1:2) and air dried atroom temperature for 15 minutes. The sheets are developed using benzene as the mobile phase. The sheets are air dried and the 1 1 major UV. and blue tetrazolium positive section is eluded with ethyl acetate. The ethyl acetate solution is washed with water, dried and concentrated to a small volume. Petroleum other is added to precipitate the 170:,21- dihydroxy-6a,16a-dimethyl-4-pregnene-3,ZO-dione.

EXAMPLE 12 Preparation of 1 15,1 7oc,2 1 -tri hydroxy-6 11,1 6 a-dim ethyl- 4-pregnene-3,20-di0rze A medium is prepared having the following composition:

Glucose g 20 An enzymatic lactoalbumen digest (Edamin) g 20 Corn steep liquor ml 5 Water to make 1 liter.

This medium is distributed in 50 ml. portions in appropriate vessels. The pH of the medium is adjusted to 6.5 with 1 M potassium hydroxide and sterilized at 120 C. for 12 minutes.

The medium in each vessel is then inoculated with an aqueous suspension of spores of the strain of Curvularia lunata (Northern Regional Research Laboratory N0. 2434) and the regulated media are maintained at an incubation temperature of 28 C. for 48 hours on a rotary shaking machine.

Ten mg. of 17a,21-dihydroxy-6u,16a-dimethyl-4-pregnene-3,20-dione prepared as described in Example 11 is added to each vessel from a dimethylformamide solution (100 mg./ml.). The incubation is carried. out for an additional 24 hours under conditions identical to the growth phase. The whole broth is then extracted three times with equal volumes of ethyl acetate, the extracts combined, and finally concentrated. The 11,3,17a,21-trihydr0xy-6a,l6adimethyl-4-pregnene-3,20-dione is filtered ofi.

The 11B,17a,21-trihydroxy-6a,16a dimethyl 4 pregnene-3,20-dione is treated with acetic anhydride and pyridine to give the 21-acetyl derivative, which is purified by recrystallization from benzene-petroleum ether to give substantially pure 115,1706,21-tl'lhYCllOXY-6t2,160t-dlmtl1- yl-4-pregnene-3,20-di0ne ZI-acetate.

EXAMPLE 13 Preparation of 1711,21-dihydroxy-6a,16oa-a'imefl1yl-4-pregnone-3 ,1 1,20-tri0ne A solution of 200 mg. of 11fi,17a,21-trihydroxy-6a,16adimethyl-4-pregnene-3,20-dione 21-acetate in 2 ml. of pyridine is added to the complex formed by the addition of 200 mg. of chromium trioxide to 2 ml. of pyridine. The mixture is swirled until thoroughly mixed and then allowed to stand at room temperature for about 16 hours. The reaction mixture is poured into water, and the aqueous mixture is extracted with ether and then twice with ethyl acetate. The combined ether and ethyl acetate extracts are washed with dilute aqueous sulfuric acid at about 0 C., and then with water until neutral. The organic solvent layer is then dried, the solvents are evaporated therefrom in vacuo, and the residual crystalline material is purified by crystallization from ethyl acetateether to give 17a,21-dihydroxy-6a,16a-dimethyl-4-pregnene-3,11,20 tri0ne 21-acetate.

Twenty-five milligrams of 17a,21-dihydroxy-6a,16a-dimethyl-4-pregnene-3,11,20-trione 21-acetate is dissolved in 1 ml. of methanol and .29 ml. of .21 N sodium methoxide in methanol is added and the solution is stirred at 20 C. under nitrogen for 7 minutes. The reaction mixture is acidified with acetic acid and diluted with water. After evaporation, of the methanol, in vacuo, the precipitate is filtered and dried. The product is crystallized from ethyl acetate-ether to give 170t,2l'dlhydlOXy-60t,160t-dl1Il6thYl-4- pregnene-3,11,20-trione.

EXAMPLE 14 Preparation of I1o,17a,21-trihydr0xy-6x,16a-dimethyl-4- pregnene-3,20-dione A medium is prepared having the following composition:

Glucose ..g 20 An enzymatic lactoalbumen digest (Edamin) g.. 20 Corn steep liquor ml 5 Water to make 1 liter.

This medium is distributed in 50 ml. portions in appro priate vessels. The pH of the medium is adjusted to 6.5 with 1 M of potassium hydroxide and sterilized at 120 C. for 12 minutes.

The medium in each vessel is then inoculated with a heavy aqueous suspension of spores of a strain of Rhizw pus nigricarzs (American Type Culture Collection No. 6227b) and the inoculated media are maintained at an incubation of 28 C. for 48 hours on a rotary shaking machine.

Ten mg. of 17u,21-dihydroxy-6ix,16a-dimethy1-4-ipregnene-3,20-dione prepared as described in Example 11 is added to each vessel from a dimethylformamide solution (100 mg./ml.). The incubation is carried out for an additional 24 hours under conditions identical to the growth phase. The whole broth is then extracted 3 times With equal volumes of ethyl acetate, the extracts combined, and finally concentrated. The 1104,17a,2l-trihydroxy-6o,16adimethyl-4-pregnene-3,20-dione is filtered off.

The 11a,17a,21-trihydroxy-6a,16u dimethyl 4 pregnene-3,20-dione is treated with acetic anhydride and pyridine to give the 21-acetyl derivative, which is purified by recrystallization from benzene-petroleum ether to give substantially pure 1l0L,1701,21-tI'lhYdTOXY-60t,16OL-dlm6llhyl- 4pregnene-3,20-di0ne ZI-acetate.

EXAMPLE 15 Preparation of 1 70,21 -dihydroxy-6o,16a-dimethyl-4-pregnene-3,11,20-trione droxy-6u,16a-dimethyl-4-pregnene-3,20-dione 21 acetate,

there is obtained 17a,21-dihydroxy-6a,16a dimethyl 4- pregame-3,1 1,20-trione.

EXAMPLE 16 Preparation of 3,20-disemicarbaz0ne of 17a,21-trihydroxy- 6a,] 6a-dimethyl-4-pregnene-3 ,1 1,20-trione To a stirred solution of 250 mg. of 17ix,21-dihydroxy- 6a,16u-dimethyl-4-pregnene-3,11,20-trione in 6.3 ml. of methanol and 3 ml. of water is added a slurry of 340 mg. of semicarbazide hydrochloride and 190 mg. of sodium bicarbonate in 1 ml. of water. The stirred mixture is refluxed under nitrogen for 4 hours. It is then cooled to 20 C. and ml. of saturated aqueous sodium chloride is added. After 3 hours at 0 C., the precipitate of the 3,20-disemicarbazone of 17,2l-dihydroxy-6e,16a-dimethyl-4-pregnene-3,11,20 trione is filtered, washed with water, until free of chloride ion and dried in air.

EXAMPLE 1'! Preparation of 3,20-disemicarbaz0ne of 11/3,17a,21-trihydroxy-6ot,1 6a-aimethyl-4-pregnene-3,20-di0ne To a stirred solution of 300 mg. of the 3,20-disemicarbazone of 1704,21- dihydroxy-6a,16a-dimethyl-4-pregnene-3,11,20-trione in 15 ml. of tetrahydrofuran and 10 ml. of water is added mg. of sodium borohydride. The stirred suspension is refluxed two hours and then cooled to 15 C. Aqueous acetic acid (3 ml. of 30%) is added cautiously and most of the tetrahydrofuran is removed in vacuo. The product crystallizes on addition of 13 ml. of 50% saturated aqueous sodium chloride and aged at 0 C. The product, 3,20-disemicarbazone of 11/3, 17a,21 trihydroxy 6m,16a dimethyl 4-pregnene-3,20- dione is filtered, washed with water and dried in air.

EXAMPLE 18 Preparation 0 f 1 15,1 711,2] -trihydr0xy-6a,1 6 a-dimethyl- 4-pregnene-3,20-di0ne To a solution of 250 mg. of the 3,20-disemicarbazone of llB,17a,2l trihydroxy 6a,16a-dimethyl-4-pregnene- 3,20-dione in 2.5 ml. of acetic acid is added .5 ml. of water and 0.25 ml. of pyruvic acid. The solution is maintained at 25 C. for 18 hours. Water (30 ml.) is added and the mixture is extracted thoroughly with chloroform. The chloroform extract is dried over sodium sulfate and taken to dryness. The residue is crystallized from ethyl acetate to give pure 11;3,l7a,21-trihydroxy-6a,l6a-dimethyl-4-pregnene-3,20-dione.

EXAMPLE 19 Preparation 0 f 1 7a,21-dihydr0xy-6a,1 6 a-dim ethy [-1 ,4- pregnadiene-3 ,1 O-dione 21 -acetate l7a,2l dihydroxy 6a,1 6a-dimethyl-4-pregnene-3,20- dione (prepared as described in Example 11) is treated with acetic anhydride and pyridine to give the 21-acetyl derivative, which is purified by recrystallization from benzene-petroleum ether to give substantially pure 17oz,2l dihydroxy-fia, 16a-dimethyl-4-pregnene-3,20-dione 21-acetate.

To 50 mg. of 17a,2l-dihydroxy-6a,16a-dimethyl-4-pregnene-3,20-dione 2l-acetate thus obtained in 2.5 ml. of t-butanol and 0.1 ml. of acetic acid is added 30 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 24 hours, 25 mg. of selenium dioxide is added and the mixture refluxed an additional 24 hours. The mixture is filtered and the filtrate taken to dryness. The residue is taken up in ethyl acetate and washed successively with aqueous sodium bicarbonate, ammonium sulfide, dilute ammonia water, water, dilute hydrochloric acid, water and is dried over magnesium sulfate. It is then treated with activated charcoal and concentrated to dryness. Crystallization of the residue from acetone-ether gives pure 170:,21 dihydroxy-6a,l6a-dimethyl-1,4-pregnadiene-3,20- dione 2l-acetate.

EXAMPLE 20 Preparation of 1 1 [3,1 7 01,21 -trihydr0xy-6a,1 6a-dimethyl- 1 ,4-pregnadiene-3 ,2 O-d ione The fermentation procedure of Example 12 is repeated but using 17a,21 dihydroxy-6a,16a-dimethyl-1,4-pregnadiene-3,20-dione 21-acetate in place of 17u,2l-dihydroxy- 6a,16a-dimethyl-4-pregnene-3,20-dione and 11B,17a,21-trihydroxy-6a,l6a-dimethy1-1,4-pregnadiene-3,20-dione is recovered.

EXAMPLE 21 Preparation of 1]ea]70:,21-trihydr0xy-6a,16a-dimethyl- 1 ,4-pregnadiene-3,20-dione Ten mg. of l7a,21-trihydroxy-6a,16u-dimethyl-l,4-pregnadiene-3,20-dione 21-acetate (obtained as in Example 19) is subjected to the action of an oxygenating enzyme produced by a strain of Rhizopus nigricans (American Type Culture Collection No. 6227b) and 11a,17a,21-trihydroxy 601,160: dimethyl-1,4-pregnadiene-3,20-dione is formed. This microbial procedure is fully described in Example 14.

EXAMPLE 22 Preparation of 1 7 a,2 1 -dihydroxy-6 [1,1 6a-dimethyl-1 ,4- pregnadiene-3,1 1,20-trione The 1 1a,17a,21-trihydroxy-6a,16a-dimethyl-1,4-pregnadiene-3,20-dione (prepared as in Example 21) is oxidized with chromium trioxide to form 17a,21-dihydroxy-6a,l6adimethyl-l,4-pregnadiene-3,11,20-trione. This procedure is fully described in Example 13.

EXAMPLE 23 Preparation of 3,20-disemicarbazone of 1 711,21 -dihydr0xy- 6a,16a-dimethyl-1,4-pregnadiene-3,11,ZO-trione To a stirred solution of 125 mg. of 17a,21-dihydroxy- 6a,16a-dimethyl-1,4-pregnadieue-3,11,20-trione (obtained as in Example 22) in 3.1 ml. of methanol and 3 ml. of water is added a slurry of 170 mg. of semicarbazide hydrochloride and'90 mg. of sodium bicarbonate in .5 ml. of water. The stirred mixture is refluxed under nitrogen for four hours. It is then cooled to 20 C. and 25 ml. of 50% saturated aqueous sodium chloride is added. After 3 hours at 0 C. the precipitate of the 3,20-disemicarbazone of 17a,21-dihydroxy-6a,1fia-dimethyl 1,4 pregnadiene- 3,11,20-trione is filtered, washed with water, until free of chloride ion and dried in air.

EXAMPLE 24 Preparation of 3,20-disemicarbazone of 11/3,17a,21-trihydr0xy-6a,1 6a-dimethyl-1,4- pregnadiene-3,2O-dione To a stirred solution of 150 mg. of the 3,20-disemicarbazone of l7a,2l-dihydroxy-6a,16a-dimethyl-1,4-pregnadiene-3,11,20-trione in 7.5 ml. of tetrahydrofuran and 5 ml. of water is added mg. of sodium borohydride. The stirred suspension is refluxed three hours and then cooled to 15 C. Aqueous acetic acid (1.5 ml. of 30%) is added cautiously and most of the tetrahydrofuran is removed in vacuo. The product crystallizes on addition of 10 ml. of 50% saturated aqueous sodium chloride and aged at 0 C. The product, 3,20-disemicarbazone of 11 3,17a,21-trihydroxy-6ot,16a-dimethyl-1,4 pregnadiene- 3,20 dione is filtered, washed with water and dried in air.

EXAMPLE 25 Preparation of 118,] 7a,21 -trihydr0xy-6 11,1 6 a-dimethyl- 1,4-pregnadiene-3,20-dione To a solution of mg. of the 3,20-disemicarbazone of l15,17a,21-trihydroxy-6a,16a-dimethyl-l,4 pregnadiene-3,20-dione in 1.2 ml. of acetic acid is added .25 ml. of water and 0.12 ml. of pyruvic acid. The solution is maintained at 25 C. for 18 hours (water 10 ml.) is added and the mixture is extracted thoroughly with chloroform. The chloroform extract is dried over sodium sulfate and taken to dryness. The residue is crystallized from ethyl acetate to give pure 1113,170:,2l-tlihYdIOXY-6a,l6oc-dimethyl-1,4- pregnadiene-3,20-dione.

Various changes and modifications may be made in the present invention, certain preferred embodiments of which are herein disclosed, Without departing from the scope thereof; to the extent that these changes and modifications are within the scope of the appended claims they are to be considered a part of this invention.

We claim:

1. 6a,16a-di-lower alkyl-17a,21-dihydroxy-4 pregnen- 3,20-dione.

2. 17a,21-dihydroxy-6a,16a-dimethyl-4-pregnen 3,20- dione.

3. A compound of the formula:

wherein R is lower alkyl and R is selected from the group consisting of hydrogen and lower alkanoyl and the dotted line between carbon 1 and 2 indicates thafia double bond 7 6 v 6 OTHER REFERENCES my f Present in this Pwition Arth et al.: so JACS 3/60 (June 1958 1 Referen'ces Cited Spero et a1.: 78 JACS 6213 (1956). UNITED STATES PATENTS Market et a1.:' 64 IACS 128.0 (1942).

2,805,230 9/1957 Stork et a1 260-3974 LEWIS GOTTS Primary Examiner 3,116,303 12/1963 Taub et a1. 260397.45

3,147,290 1 9/1964 Spero 260-39146 B T L. ROBERTS, Examin r. 3,176,032 3/1965 Ringold et a1. 260397.47

3,201,429 8/1965 Djerassi et a1. 260397.47 10 T. M. MESHBESHER, Assistant Examiner. 

3. A COMPOUND OF THE FORMULA: 